Nature-inspired pyrrolo[2,3-d]pyrimidines targeting the histamine H3 receptor

Article abstract of DOI:10.1016/j.bmc.2019.05.042

Inspired by marine compounds the derivatization of the natural pyrrolo[2,3-d]pyrimidine lead scaffold led to a series of novel compounds targeting the histamine H₃ receptor. The focus was set on improved binding towards the receptor and to esta

Full text of DOI:10.1016/j.bmc.2019.05.042

Accepted Manuscript
Nature-Inspired Pyrrolo[2,3-d]pyrimidines Targeting the Histamine H3 Recep-
tor
Annika Frank, Francisco Meza-Arriagada, Cristian O. Salas, Christian
Espinosa-Bustos, Holger Stark
PII:
S0968-0896(19)30431-6
https://doi.org/10.1016/j.bmc.2019.05.042
BMC 14929
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
15 March 2019
5 April 2019
28 May 2019
Please cite this article as: Frank, A., Meza-Arriagada, F., Salas, C.O., Espinosa-Bustos, C., Stark, H., Nature-Inspired
Pyrrolo[2,3-d]pyrimidines Targeting the Histamine H3 Receptor, Bioorganic & Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.bmc.2019.05.042
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Nature-Inspired Pyrrolo[2,3-d]pyrimidines Targeting the Histamine H3 Receptor
Annika Franka, Francisco Meza-Arriagadab, Cristian O. Salasc, Christian Espinosa-Bustosb*, Holger
Starka*
aInstitute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf,
Universitaetsstr. 1, 40225 Duesseldorf, Germany; E-mail: stark@hhu.de
bDepartamento de Farmacia, Facultad de Química y de Farmacia, Pontificia Universidad Católica de
Chile, Santiago 6094411, Chile; E-mail: ccespino@uc.cl
cDepartamento de Química Orgánica, Facultad de Química y de Farmacia, Pontificia Universidad
Católica de Chile, Santiago 6094411, Chile
dedicated to Prof. Peter Proksch on his 65th birthday
Keywords: Heterocycles, Histamine, G-protein coupled receptor, Natural products, H3R, Microwave-
assisted synthesis
Running title: Nature Inspired Compounds at Histamine H3R
1

Abstract:
Inspired by marine compounds the derivatization of the natural pyrrolo[2,3-d]pyrimidine lead
scaffold led to a series of novel compounds targeting the histamine H3 receptor. The focus was set on
improved binding towards the receptor and to establish an initial structure-activity relationship for this
compound class based on the lead structure (compound V, Ki value of 126 nM). As highest binding
affinities were found with 1,4-bipiperidines as basic part of the ligands, further optimization was
focused on 4-([1,4'-bipiperidin]-1'-yl)-pyrrolo[2,3-d]pyrimidines. Related pyrrolo[2,3-d]pyrimidines
that were isolated from marine sponges like 4-amino-5-bromopyrrolo[2,3-d]pyrimidine
(compound III), showed variations in halogenation pattern, though in a next step the impact of
halogenation at 2-position was evaluated. The chloro variations did not improve the affinity compared
to the dehalogenated compounds. However, the simultaneous introduction of lipophilic cores with
electron-withdrawing substitution patterns in 7-position and dehalogenation at 2-position (11b, 12b)
resulted in compounds with significantly higher binding affinities (Ki values of 7 nM and 6 nM,
respectively) than the initial lead structure compound V. The presented structures allow for a
reasonable structure-activity relationship of pyrrolo[2,3-d]pyrimidines as histamine H3 receptor
ligands and yielded novel lead structures within the natural compound library against this target.
2

1 Introduction
Natural products are a highly appreciated source for novel lead compounds, as they are pre-
designed for biological activity by natural optimization and provide with large structural heterogeneity
high hit rates in pharmacological screenings.1 A remarkable amount of currently used therapeutics are
natural compounds or are at least inspired by nature (e.g. antibiotic or cytostatic agents).2 The
combination of high hit rates and a given drug-likeness motivates researchers to re-investigate
compounds, already applied for a certain application field, for the use in other areas as well. One of
the most investigated drug targets to date, is the class of G-protein coupled receptors (GPCRs). Among
these, the four subtypes of the histamine receptor represent a highly diverse target class as the
different receptors are involved in allergy, gastric acid secretion, inflammation or even
neurodegenerative diseases.3 Recent research on the histamine H3 receptor (H3R) emphasizes its
crucial role in the central nervous system. The presynaptic H3 autoreceptors modulate histamine
release in the central nervous system and due to its heterodimerization with non-histaminergic
receptors also a variety of other neurotransmitters such as dopamine, acetylcholine or noradrenaline.4
Owing to its involvement in the pathophysiology of Alzheimer´s disease, attention deficit hyperactivity
disorder and schizophrenia (amongst others), it became apparent that targeting the H3R may reduce
the cognitive impairments of those diseases.5 The search for agents targeting the receptor led to the
investigation of two natural ligands, that were found as novel potential lead structures.4 Compound I
(Figure 1, Ki value of 30 nM) was isolated from the marine sponge Aplysina sp. and inspired the design
of a variety of novel non-imidazole-based ligands.6 Derivatization of the basic moiety and the spacer
length of the bromotyrosin derivative led to the discovery of non-imidazole compounds like pitolisant
(Figure 2) that display high activity and optimized scaffolds towards the H3R. In a similar manner,
various modifications were performed with the natural compound conessine7 (compound II, Figure 1)
that was also found to be an H3R antagonist. One attempt aimed for rigidizing the alkaloid, while others
attempted to aromatize and simplify the scaffold, resulting in H3R antagonists like compound III (Figure
2), displaying affinities in the low nanomolar ranges.8
3

Figure 1: Natural compounds targeting the H3R and inspired the presented drug design approach.
Although the compound library for targeting the H3R is increasing rapidly, to date, only one ligand
passed clinical evaluation and entered the market. Pitolisant, an inverse H3R agonist has been
approved for the treatment of narcolepsy by the EMA in 2016 and is currently undergoing clinical phase
II and III studies for cognitive enhancement in patients with schizophrenia or Parkinson´s diesease.9,10
The ligand adheres to the typical blueprint of H3R ligands (Figure 2). It consists of a basic aliphatic
amine moiety (blue), responsible for binding to the receptor, and a linker, most often an alkyl linker,
connecting the amine to the central core (red). Through a second linker the core is connected to an
arbitrary, lipophilic region (green), which aids in improving selectivity.11 As shown with compounds III
and pitolisant the derivatization of natural products can lead to novel compounds, adhering to this
blueprint, with promising activities at the H3R. Though the drug research community at GPCRs is in
constant search for novel scaffolds inspired by Mother Nature.
4

Figure 2: General blueprint for H3R ligands and structures of pitolisant and the lead structure. Blue: basic amine, red: central
core, green: arbitrary region.
Pyrrolo[2,3-d]pyrimidines are a class of natural compounds that were found in several marine
organisms, like sponges or algae. The brominated analogue compound IV (Figure 1) for example, was
isolated from Echinodictyum sp. and is a promising inhibitor of the adenosine kinase.12 The compound
class is known to provide promising results in anti-inflammatory and anti-infectious assays in various
studies.13,14 As the pathophysiology of neurodegenerative diseases involves inflammatory events too,
multiple treatment strategies focus on the anti-inflammatory properties of novel substances.15 H3R
antagonists are reported to improve cognitive functions in neurodegenerative diseases,5 though the
synergistic effect of targeting the H3R and the known anti-inflammatory properties of pyrrolo[2,3-
d]pyrimidines13 may aid current drug development of neurological diseases. Though we recently
conducted the combination of the natural pyrrolo[2,3-d]pyrimidines with the general pharmacophore
of H3R ligands to expand possible application fields towards neurodegenerative diseases.16 The novel
ligands showed selectivity for the H3R compared to the H4R and displayed promising structure-activity
relationships (SARs). Among the tested derivatives, a 1,4-bipiperidine warhead as the basic moiety of
the molecule was superior to morpholines or piperazines regarding its affinity towards the H3R, with
the most active compound (compound V, Figure 1) displaying a Ki value of 126 nM. Based on these
results we went for a rationalized drug optimization approach to improve pharmacological activity of
the novel scaffold. As morpholines are frequently used in the design of H3R ligands,17 the first
derivatization introduced morpholines as basic group (Y, Figure 3) and into the lipophilic core at 7-
position of the pyrrolo[2,3-d]pyrimidine (R, Figure 3). A second focus was set on the impact of
halogenation at 2-position (X, Figure 3), as pyrrolo[2,3-d]pyrimidines isolated from marine organisms
5

are typically halogenated, as seen with compound IV. To complete the SAR investigation R was
derivatized by introducing varying lipophilic residues. All compounds were tested for their H3R binding
affinities and screened for selectivity towards the H4R. The goal of this study was to optimize the
existing pharmacophore of pyrrolo[2,3-d]pyrimidines and to increase H3R binding affinity compared to
compound V. In doing so, the class of H3R ligands can be expanded by natural inspired compounds that
may facilitate the jump from bench to bedside.
Figure 3: Possible derivatization strategies of the initial lead structure.
2 Material and Methods
2.1 Materials
Melting points were determined on a Kofler Thermogerate apparatus and were uncorrected.
All reagents were purchased from Sigma-Aldrich, unless otherwise specified. The NMR spectra were
recorded on NMR Bruker AV 400. Chemical shifts were given in parts per million relative to TMS [1H
and 13C, δ(SiMe4) = 0]. Most NMR assignments were supported by additional 2D experiments. HRMS-
ESI-MS experiments were carried out using a Thermo Scientific Exactive Plus Orbitrap Spectrometer.
Thin layer chromatography (TLC) was performed using Merck GF-254 type 60 silica gel. Column
chromatography was carried out using Merck silica gel 60 (70–230 mesh). Radioligands [3H]Nα-
methylhistamine and [3H]histamine were purchased at PerkinElmer. HEK-293 cells stably expressing
the human H3 receptor were kindly gifted by Prof. Dr. Jean-Charles Schwartz (Bioprojet, France). Sf9-
hH4-Gαi2-Gβ1γ2 cells were a kind donation by Prof. Dr. Seifert.
2.2 Methods
2.2.1 Chemical Part
6

For the synthesis of pyrrolo[2,3-d]pyrimidin-7-yl-ethylmorpholine derivatives 3a-c and 4a-c, a simple
synthetic strategy was used (Scheme 1). Pyrrolo[2,3-d]pyrimidines 1a-c (1 eq.), 2-
chloroethylmorpholine (2 eq.) and potassium carbonate (3 eq.) were mixed in acetonitrile and the
reaction mixture was stirred at 90 °C for 6 h, obtaining the corresponding 2a-c derivatives.
Subsequently, the target compounds were obtained through a microwave-assisted aromatic
nucleophilic substitution reaction. The bipiperidine and morpholino-piperidine scaffolds were
incorporated in the 4-position of the heterocycle using triethylamine and ethanol as the solvent.
The derivatives 9a-12a and 9b-12b were synthesized from the pyrrolopyrimidines 1a-b according to
scheme 1. In a first synthetic step, an N-alkylation reaction was performed using various benzyl halides
in acetonitrile under reflux conditions and then mediated by microwave-assisted conditions the
bipiperidine fragment was added.
7

Scheme 1: Reagents and conditions. a) 2-(Chloroethyl)morpholine, K2CO3, MeCN, reflux, 6 h. b) Bipiperidine, EtOH, NEt3,
80°C, mw, 15 min. c) 4-(Piperidin-4-yl)morpholine, EtOH, NEt3, 80°C, mw, 15 min. d) Benzyl halides, K2CO3, CH3CN, reflux, 3 h.
e) Bipiperidine, EtOH, NEt3, 80°C, mw, 15 min.
2.2.2 Chemistry
2.2.2.1 General Synthetic Procedure to Obtain 4-(2-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-7-
yl)ethyl)morpholine Derivatives 2a-c.
8

A mixture of the corresponding pyrrolopyrimidine (1.0 mmol), 4-(2-chloroethyl)morpholine (2.0 mmol)
and potassium carbonate (3.0 mmol) in acetonitrile (5 mL) was stirred for 6 h, then the mixture was
filtered and evaporated under vacuum. The products were separated by flash chromatography on silica
gel eluting with methanol/methylene chloride 1:20.
4-(2-(2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)morpholine 2a. Light brown solid, yield 76
%, Mp 79-80 °C. 1H NMR (400 MHz, CDCl3) δ 6.91 (d, J = 3.6 Hz, 1H, H6), 6.33 (d, J = 3.6 Hz, 1H, H5),
4.13 (t, J = 6.5 Hz, 2H, 2 x H1’), 3.67 – 3.57 (m, 4H, 2 x H5’ and 2 x H7’), 2.67 (t, J = 6.5 Hz, 2H, 2 x H2’),
2.53 – 2.43 (m, 4H, 2 x H4’ and 2 x H8’). 13C NMR (101 MHz, CDCl3) δ 158.42, 153.41, 152.52, 126.24,
110.66, 99.49, 66.94 (2C), 57.94, 53.66 (2C), 41.67.
4-(2-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)morpholine 2b. Brown solid, yield 77 %, Mp
126-128 °C. 1H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H, H2), 7.32 (d, J = 3.6 Hz, 1H, H6), 6.54 (d, J = 3.6 Hz,
1H, H5), 4.34 (t, J = 6.3 Hz, 2H, 2 x H1’), 3.65 – 3.56 (m, 4H, 2 x H5’ and 2 x H7’), 2.72 (t, J = 6.3 Hz, 2H,
2 x H2’), 2.51 – 2.39 (m, 4H, 2 x H4’ and 2 x H8’). 13C NMR (101 MHz, CDCl3) δ 162.48, 151.96, 151.03,
150.43, 129.73, 117.42, 99.25, 66.88 (2C), 58.04, 53.57 (2C), 42.02.
4-Chloro-7-(2-morpholinoethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine 2c. White solid, yield 75 %, Mp
133-135 °C. 1H NMR (400 MHz, CDCl3) δ 6.91 (d, J = 3.6 Hz, 1H, H6), 6.33 (d, J = 3.6 Hz, 1H, H5), 5.04 (s,
2H, NH2), 4.13 (t, J = 6.5 Hz, 2H, 2 x H1’), 3.69 – 3.50 (m, 4H, 2 x H5’ and 2 x H7’), 2.67 (t, J = 6.5 Hz, 2H,
2 x H2’), 2.51 – 2.40 (m, 4H, 2 x H4’ and 2 x H8’). 13C NMR (101 MHz, CDCl3) δ 158.45, 153.41, 152.51,
126.23, 110.63, 99.48, 66.94, 57.94, 53.66, 41.67.
2.2.2.2 General Synthetic Procedure to Obtain 4-(2-(4-([1,4'-Bipiperidin]-1'-yl)-7H-pyrrolo[2,3-
d]pyrimidin-7-yl)ethyl)morpholine Derivatives 3a-c and 4a-c
Compounds 2a-c and (1.0 mmol), 4-piperidinopiperidine or 4-(piperidin-4-yl)morpholine (3.0 mmol),
triethylamine (4.5 mmol) and ethanol (5 mL) were added to a microwave reaction flask and the
reaction mixture was irradiated for 15 min at 80 °C. Then the solvent was evaporated under vacuum
and the crude product was purified by column chromatography on silica gel using
chloroform/methanol (10:1) mixture.
9

4-(2-(2-Chloro-4-(4-morpholinopiperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)morpholine 3a.
Brown oil, yield 95 %. 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 3.6 Hz, 1H, H6), 6.40 (d, J = 3.7 Hz, 1H,
H5), 4.72 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 4.21 (t, J = 6.3 Hz, 2H, 2 x H1’), 3.71 – 3.67 (m, 4H, 2 x H9’’
and 2 x H11’’), 3.66 – 3.62 (m, 4H, 2 x H5’ and 2 x H7’), 3.14 – 3.00 (m, 2H, H2’’ and H6’’), 2.68 (t, J =
6.3 Hz, 2H, 2 x H2’), 2.56 – 2.52 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.51 – 2.43 (m, 5H, 2 x H4’, 2 x H8’ and
H4’’), 1.95 (d, J = 12.8 Hz, 2H, H3’’ and H5’’), 1.54 (ddd, J = 15.9, 12.5, 4.1 Hz, 2H, H3’’ and H5’’). 13C
NMR (101 MHz, CDCl3) δ 157.13, 153.17, 152.35, 124.13, 101.24, 100.92, 67.25 (2C), 66.98 (2C), 61.88,
58.19, 53.65 (2C), 49.78 (2C), 45.13 (2C), 41.68, 28.24 (2C). HRMS m/z 435.2270 [M+H+] (calcd. for
C21H31ClN6O2, 435.2201).
4-(1-(7-(2-Morpholinoethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-yl)morpholine 3b. Light
brown solid, yield 65 %, Mp 114-115 °C. 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H, H2), 6.99 (d, J = 3.6 Hz,
1H, H6), 6.43 (d, J = 3.6 Hz, 1H, H5), 4.75 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 4.27 (t, J = 6.5 Hz, 2H, 2 x
H1’), 3.71 – 3.66 (m, 4H, 2 x H9’’ and 2 x H11’’), 3.66 – 3.61 (m, 4H, 2 x H5’ and 2 x H7’), 3.12 – 3.01 (m,
2H, H2’’ and H6’’), 2.71 (t, J = 6.5 Hz, 2H, 2 x H2’), 2.57 – 2.52 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.52 – 2.43
(m, 5H, 2 x H4’, 2 x H8’ and H4’’), 1.94 (d, J = 11.4 Hz, 2H, H3’’ and H5’’), 1.55 (ddd, J = 24.1, 12.3, 4.1
Hz, 2H, H3’’ and H5’’). 13C NMR (101 MHz, CDCl3) δ 156.87, 151.19, 151.14, 123.87, 103.08, 100.59,
67.29 (2C), 66.98 (2C), 62.16, 58.22, 53.68 (2C), 49.78 (2C), 45.24 (2C), 41.80, 28.29 (2C). HRMS m/z
401.2660 [M+H+] (calcd. for C21H32N6O2, 401.2599).
7-(2-Morpholinoethyl)-4-(4-morpholinopiperidin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine 3c. Light
brown solid, yield 22 %, Mp 153-155 °C. 1H NMR (400 MHz, CDCl3) δ 6.70 (d, J = 3.6 Hz, 1H, H6), 6.29
(d, J = 3.6 Hz, 1H, H5), 4.70 (d, J = 13.2 Hz, 2H, H2’’ and H6’’), 4.52 (s, 2H, NH2), 4.11 (t, J = 6.7 Hz, 2H, 2
x H1’), 3.71 – 3.65 (m, 8H, 2 x H5’, 2 x H7’, 2 x H9’’ and 2 x H11’’), 2.97 (t, J = 12.0 Hz, 2H, H2’’ and H6’’),
2.67 (t, J = 6.7 Hz, 2H, 2 x H2’), 2.56 – 2.49 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.48 – 2.41 (m, 5H, 2 x H4’, 2
x H8’ and H4’’), 1.89 (d, J = 11.7 Hz, 2H, H3’’ and H5’’), 1.51 (ddd, J = 24.2, 12.2, 3.9 Hz, 2H, H3’’ and
H5’’). 13C NMR (101 MHz, CDCl3) δ 158.72, 157.62, 153.62, 121.03, 100.97, 97.13, 67.30 (2C), 66.98
10

(2C), 62.29, 58.13, 53.71 (2C), 49.74 (2C), 45.06 (2C), 41.46, 28.23 (2C). HRMS m/z 416.2768 [M+H+]
(calcd. for C21H33N7O2, 416.2708).
4-(2-(4-([1,4'-Bipiperidin]-1'-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)morpholine
4a.
Yellow oil, yield 87 %. 1H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 3.6 Hz, 1H, H6), 6.40 (d, J = 3.7 Hz, 1H,
H5), 4.76 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 4.21 (t, J = 6.3 Hz, 2H, 2 x H1’), 3.65 – 3.61 (m, 4H, 2 x H5’
and 2 x H7’), 3.08 – 2.98 (m, 2H, H2’’ and H6’’), 2.67 (t, J = 6.3 Hz, 2H, 2 x H2’), 2.64 – 2.58 (m, 1H, H4’’),
2.57 – 2.51 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.48 – 2.43 (m, 4H, 2 x H4’, 2 x H8’), 1.96 (d, J = 12.0 Hz, 2H,
H9’’ and H11’’), 1.65 – 1.52 (m, 6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.46 – 1.38 (m, 2H, 2 x H10’’). 13C
NMR (101 MHz, CDCl3) δ 157.09, 153.16, 152.34, 124.11, 101.22, 100.95, 66.98 (2C), 62.67, 58.19,
53.65 (2C), 50.17 (2C), 45.51 (2C), 41.68, 27.79 (2C), 26.00 (2C), 24.52. HRMS m/z 433.2477 [M+H+]
(calcd. for C22H33ClN6O, 433.2418).
4-(2-(4-([1,4'-Bipiperidin]-1'-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)morpholine 4b. Brown solid,
yield 72 %, Mp 110-112 °C. 1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H, H2), 7.02 (d, J = 3.6 Hz, 1H, H6),
6.44 (d, J = 3.6 Hz, 1H, H5), 4.84 (d, J = 13.3 Hz, 2H, 2 x H2’’), 4.29 (t, J = 6.5 Hz, 2H, 2 x H1’), 3.71 – 3.60
(m, 4H, 2 x H5’ and 2 x H7’), 3.06 (t, J = 12.4 Hz, 2H, H2’’ and H6’’), 2.84 (t, J = 10.9 Hz, 1H, H4’’), 2.76 –
2.61 (m, 6H, 2 x H2’, 2 x H8’’ and 2 x H12’’), 2.52 – 2.46 (m, 4H, 2 x H4’, 2 x H8’), 2.09 (d, J = 11.9 Hz,
2H, H9’’ and H11’’), 1.81 – 1.60 (m, 6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.49 (br, 2H, 2 x H10’’). 13C
NMR (101 MHz, CDCl3) δ 156.72, 151.22, 151.06, 124.08, 103.10, 100.48, 66.97 (2C), 63.31, 58.20,
53.67 (2C), 50.05 (2C), 45.35 (2C), 41.80, 27.33 (2C), 25.17 (2C), 24.01. HRMS m/z 399.2867 [M+H+]
(calcd. for C22H34N6O, 399.2811).
4-([1,4'-Bipiperidin]-1'-yl)-7-(2-morpholinoethyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine 4c. White
solid, yield 39 %, Mp 146-148 °C. 1H NMR (400 MHz, CDCl3) δ 6.68 (d, J = 3.6 Hz, 1H, H6), 6.27 (d, J =
3.6 Hz, 1H, H5), 4.73 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 4.57 (s, 2H, NH2), 4.09 (t, J = 6.7 Hz, 2H, 2 x H1’),
3.68 – 3.61 (m, 4H, 2 x H5’ and 2 x H7’), 2.92 (t, J = 12.1 Hz, 2H, 2 x H2’), 2.71 – 2.61 (m, 3H, H2’’, H4’’
and H6’’), 2.60 – 2.54 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.48 – 2.43 (m, 4H, 2 x H4’, 2 x H8’), 1.94 (d, J =
12.0 Hz, 2H, H9’’ and H11’’), 1.68 – 1.50 (m, 6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.50 – 1.33 (m, 2H, 2
x H10’’). 13C NMR (101 MHz, CDCl3) δ 158.72, 157.52, 153.55, 121.09, 100.96, 97.10, 66.95 (2C), 63.20,
58.09, 53.69 (2C), 50.06 (2C), 45.30 (2C), 41.51, 27.53 (2C), 25.62 (2C), 24.31. HRMS m/z 414.2976
[M+H+] (calcd. for C22H35N7O, 414.2914).
2.2.2.3 General Synthetic Procedure to Obtain N-Benzyl-pyrrolo[2,3-d]pyrimidine Derivatives 5-8a and
5-8b.
11

A mixture of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine or 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine
(1.0 mmol), the respective benzyl halide (1.2 mmol) and potassium carbonate (3.0 mmol) in
acetonitrile (5 mL) was stirred for 3 h, then the mixture was filtered and evaporated under vacuum.
The products were separated by flash chromatography on silica gel eluting with methylene chloride.
2,4-Dichloro-7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 5a. White solid, yield 72 %, Mp 129-131
1
°C. H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.4 Hz, 2H, H3’ and H5’), 7.12 – 7.07 (m, 3H, H6, H2’ and
H6’), 6.55 (d, J = 3.6 Hz, 1H, H5), 5.31 (s, 2H, N-CH2). 13C NMR (101 MHz, CDCl3) δ 152.84, 152.21,
152.11, 134.41, 134.18, 129.40, 129.24 (2C), 129.15 (2C), 116.30, 100.73, 47.93. HRMS m/z 311.9857
[M+H+] (calcd. for C13H8Cl3N3, 311.9814).
2,4-Dichloro-7-(4-fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 6a. White solid, yield 67 %, Mp 96-98 °C.
1H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 8.4, 5.3 Hz, 2H, H3’ and H5’), 7.37 (d, J = 3.6 Hz, 1H, H6), 7.23
(t, J = 8.6 Hz, 2H, H2’ and H6’), 6.81 (d, J = 3.6 Hz, 1H, H5), 5.58 (s, 2H, N-CH2). 13C NMR (101 MHz, CDCl3)
δ 162.65 (d, JCF = 247.6 Hz), 152.78, 152.14, 152.06, 131.53 (d, JCF = 3.3 Hz), 129.68 (d, JCF = 8.3 Hz, 2C),
129.41, 116.31, 116.01 (d, JCF = 21.7 Hz, 2C), 100.63, 47.91. HRMS m/z 296.0152 [M+H+] (calcd. for
C13H8Cl2FN3, 296.0111).
4-((2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzonitrile 7a. White solid, yield 56 %. Mp
175-177 °C. 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 8.2 Hz, 2H, H3’ and H5’), 7.22 (d, J = 8.1 Hz, 2H, H2’
and H6’), 7.13 (d, J = 3.6 Hz, 1H, H6), 6.58 (d, J = 3.6 Hz, 1H, H5), 5.41 (s, 2H, N-CH2). 13C NMR (101 MHz,
CDCl3) δ 153.04, 152.37, 152.18, 140.98, 132.82 (2C), 129.46, 128.15 (2C), 118.22, 116.31, 112.36,
101.14, 48.08. HRMS m/z 303.0199 [M+H+] (calcd. for C14H8Cl2N4, 303.0155).
2,4-Dichloro-7-(2,6-dichlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 8a. White solid, yield 64 %, Mp 140-
142 °C. 1H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.0 Hz, 2H, H3’ and H5’), 7.14 (dd, J = 8.7, 7.3 Hz, 1H,
H4’), 6.83 (d, J = 3.7 Hz, 1H, H6), 6.38 (d, J = 3.7 Hz, 1H, H5), 5.52 (s, 2H, N-CH2). 13C NMR (101 MHz,
CDCl3) δ 152.56, 152.10, 151.94, 136.90, 130.93 (2C), 130.70, 128.90 (2C), 128.45, 116.18, 100.44,
43.67. HRMS m/z 345.9467 [M+H+] (calcd. for C13H7Cl4N3, 345.9421).
4-Chloro-7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 5b. White solid, yield 46 %, Mp 144-145 °C.
1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H, H2), 7.38 – 7.33 (m, 2H, H3’ and H5’), 7.26 (d, J = 3.6 Hz, 1H,
H6), 7.22 (d, J = 7.6 Hz, 2H, H2’ and H6’), 6.70 (d, J = 3.6 Hz, 1H, H5), 5.49 (s, 2H, N-CH2). 13C NMR (101
12

MHz, CDCl3) δ 152.37, 151.14, 151.00, 134.78, 134.19, 129.16 (2C), 128.98 (2C), 128.83, 117.51, 100.26,
47.84. HRMS m/z 278.0246 [M+H+] (calcd. for C13H9Cl2N3, 278.0210).
4-Chloro-7-(4-fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 6b. White solid, yield 56 %, Mp 103-105 °C
1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H, H2), 7.27 – 7.22 (m, 3H, H6, H3’ and H5’), 7.07 – 7.00 (m, 2H,
H2’ and H6’), 6.65 (d, J = 3.6 Hz, 1H, H5), 5.45 (s, 2H, N-CH2). 13C NMR (101 MHz, CDCl3) δ 162.53 (d, JCF
= 247.2 Hz), 152.28, 151.08, 150.92, 132.11 (d, JCF = 3.3 Hz), 129.46 (d, JCF = 8.3 Hz, 2C), 128.84, 117.50,
115.89 (d, JCF = 21.7 Hz, 2C), 100.13, 47.80. HRMS m/z 262.0542 [M+H+] (calcd. for C13H9ClFN3,
262.0507).
4-((4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzonitrile 7b. White solid, yield 59 %, Mp
157-159 °C. 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H, H2), 7.55 (d, J = 8.2 Hz, 2H, H3’ and H5’), 7.22 (d, J
= 8.1 Hz, 2H, H2’ and H6’), 7.16 (d, J = 3.6 Hz, 1H, H6), 6.61 (d, J = 3.6 Hz, 1H, H5), 5.46 (s, 2H, N-CH2).
13C NMR (101 MHz, CDCl3) δ 152.60, 151.18 (2C), 141.55, 132.77 (2C), 128.80, 127.99 (2C), 118.28,
117.53, 112.23, 100.69, 48.04. HRMS m/z 269.0589 [M+H+] (calcd. for C14H9ClN4, 269.0552).
4-Chloro-7-(2,6-dichlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 8b. White solid, yield 51 %, Mp 151-153
°C 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H, H2), 7.24 (d, J = 8.1 Hz, 2H, H3’ and H5’), 7.18 – 7.06 (m, 1H,
H4’), 6.87 (d, J = 3.7 Hz, 1H, H6), 6.40 (d, J = 3.7 Hz, 1H, H5), 5.59 (s, 2H, N-CH2). 13C NMR (101 MHz,
CDCl3) δ 152.09, 151.15, 150.76 (2C), 136.89, 131.25, 130.71, 128.85 (2C), 127.81, 117.32, 100.02,
43.42. HRMS m/z 311.9857 [M+H+] (calcd. for C13H8Cl3N3, 311.9815).
2.2.2.4 General Synthetic Procedure to Obtain Substituted Pyrrolo[2,3-d]pyrimidines 9a-12a and
9b-12b.
The N-benzyl-pyrrolo[2,3-d]pyrimidine 5-8a or 5-8b (1.0 mmol), 4-piperidinopiperidine (3.0 mmol),
triethylamine (4.5 mmol) and ethanol (5 mL) were added to a microwave reaction flask and the
reaction mixture was irradiated for 15 minutes at 80 °C. Then the solvent was evaporated under
vacuum and the crude product was purified by column chromatographic on silica gel using
chloroform/methanol (10:1) mixture.
13

4-([1,4'-Bipiperidin]-1'-yl)-2-chloro-7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 9a. White solid,
yield 53 %, Mp 97-99 °C. 1H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 7.9 Hz, 2H, H3’ and H5’), 7.09 (d, J =
8.2 Hz, 2H, H2’ and H6’), 6.77 (d, J = 3.5 Hz, 1H, H6), 6.43 (d, J = 3.6 Hz, 1H, H5), 5.26 (s, 2H, CH2), 4.78
(d, J = 13.2 Hz, 2H, H2’’ and H6’’), 3.05 (t, J = 12.5 Hz, 2H, H2’’ and H6’’), 2.72 (t, J = 11.3 Hz, 1H, H4’’),
2.60 (br, 4H, 2 x H8’’ and 2 x H12’’), 2.02 (d, J = 12.1 Hz, 2H. H9’’ and H11’’), 1.73 – 1.53 (m, 6H, 2 x H3’’,
2 x H5’’, H9’’ and H11’’), 1.45 (d, J = 4.6 Hz, 2H, 2 x H10’’). 13C NMR (101 MHz, CDCl3) δ 157.09, 153.56,
152.54, 135.47, 133.76, 129.05 (2C), 128.94 (2C), 123.28, 101.91, 101.22, 62.80, 50.14 (2C), 47.34,
45.41 (2C), 27.56 (2C), 25.62 (2C), 24.27. HRMS m/z 444.1716 [M+H+] (calcd. for C23H28Cl2N5, 444.1657).
4-([1,4'-Bipiperidin]-1'-yl)-2-chloro-7-(4-fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 10a. White solid,
yield 57 %, Mp 129-130 °C. 1H NMR (400 MHz, CDCl3) δ 7.17 (dd, J = 8.3, 5.4 Hz, 2H, H3’ and H5’), 6.98
(t, J = 8.6 Hz, 2H, H2’ and H6’), 6.79 (d, J = 3.6 Hz, 1H, H6), 6.44 (d, J = 3.7 Hz, 1H, H5), 5.28 (s, 2H, CH2),
4.79 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 3.06 (t, J = 12.1 Hz, 2H, H2’’ and H6’’), 2.73 – 2.64 (m, 1H, H4’’),
2.61 – 2.53 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.00 (d, J = 12.2 Hz, 2H, H9’’ and H11’’), 1.70 – 1.55 (m, 6H,
2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.50 – 1.37 (m, 2H, 2 x H10’’). 13C NMR (101 MHz, CDCl3) δ 162.37 (d,
JCF = 246.5 Hz), 157.11, 153.53, 152.48, 132.77 (d, JCF = 3.3 Hz), 129.47 (d, JCF = 8.2 Hz, 2C), 123.22,
115.65 (d, JCF = 21.6 Hz, 2C), 101.85, 101.21, 62.70, 50.16 (2C), 47.29, 45.47 (2C), 27.70 (2C), 25.86 (2C),
24.42. 19F NMR (376 MHz, CDCl3) δ -114.37. HRMS m/z 428.2012 [M+H+] (calcd. for C23H27ClFN5,
428.1961).
4-((4-([1,4'-Bipiperidin]-1'-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzonitrile
11a.
1
White solid, yield 77 %, Mp 158-160 °C. H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.2 Hz, 2H, H3’ and
H5’), 7.22 (d, J = 8.1 Hz, 2H, H2’ and H6’), 6.82 (d, J = 3.7 Hz, 1H, H6), 6.49 (d, J = 3.7 Hz, 1H, H5), 5.37
(s, 2H, CH2), 4.79 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 3.08 (t, J = 12.0 Hz, 2H, H2’’ and H6’’), 2.77 – 2.66
(m, 1H, H4’’), 2.66 – 2.53 (m, 4H, 2 x H8’’ and 2 x H12’’), 2.03 (d, J = 12.1 Hz, 2H, H9’’ and H11’’), 1.72
– 1.61 (m, 6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.46 (d, J = 5.0 Hz, 2H, 2 x H10’’). 13C NMR (101 MHz,
CDCl3) δ 157.08, 153.71, 152.59, 142.41, 132.56 (2C), 127.97 (2C), 123.22, 118.50, 111.71, 102.38,
101.18, 62.66, 50.16 (2C), 47.51, 45.41 (2C), 27.65 (2C), 25.71 (2C), 24.31. HRMS m/z 435.2058 [M+H+]
(calcd. for C24H27ClN6, 435.1999).
14

4-([1,4'-Bipiperidin]-1'-yl)-2-chloro-7-(2,6-dichlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 12a. White
solid, yield 65 %, Mp 94-95 °C. 1H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 8.0 Hz, 2H, H3’ and H5’), 7.15 –
7.11 (m, 1H, H4’), 6.42 (d, J = 3.7 Hz, 1H, H6), 6.23 (d, J = 3.7 Hz, 1H, H5), 5.46 (s, 2H, CH2), 4.66 (d, J =
13.3 Hz, 2H, H2’’ and H6’’), 2.92 (t, J = 12.1 Hz, 2H, H2’’ and H6’’), 2.58 (t, J = 10.9 Hz, 1H, H4’’), 2.47
(br, 4H, 2 x H8’’ and 2 x H12’’), 1.88 (d, J = 12.2 Hz, 2H, H9’’ and H11’’), 1.63 – 1.43 (m, 6H, 2 x H3’’, 2 x
H5’’, H9’’ and H11’’), 1.36 – 1.22 (m, 2H, 2 x H10’’). 13C NMR (101 MHz, CDCl3) δ 157.06, 153.31, 152.52
(2C), 137.04, 131.58, 130.44, 128.72 (2C), 121.99, 101.48, 101.20, 62.82, 50.12 (2C), 45.42, 43.39 (2C),
27.56 (2C), 25.69 (2C), 24.32. HRMS m/z 478.1327 [M+H+] (calcd. for C23H26Cl3N5, 478.1265).
4-([1,4'-Bipiperidin]-1'-yl)-7-(4-chlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 9b. Brown solid, yield 47
%, Mp 103-104 °C. 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H, H2), 7.36 (d, J = 8.1 Hz, 2H, H3’ and H5’),
7.22 (d, J = 8.0 Hz, 2H, H2’ and H6’), 7.00 (d, J = 3.1 Hz, 1H, H6), 6.60 (d, J = 3.2 Hz, 1H, H5), 5.45 (s, 2H,
CH2), 4.97 (d, J = 13.0 Hz, 2H, H2’’ and H6’’), 3.19 (t, J = 12.5 Hz, 2H, H2’’ and H6’’), 3.00 (t, J = 11.1 Hz,
1H, H4’’), 2.83 (br, 4H, 2 x H8’’ and 2 x H12’’), 2.22 (d, J = 11.6 Hz, 2H, H9’’ and H11’’), 1.94 – 1.77 (m,
6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.61 (s, 2H, 2 x H10’’). 13C NMR (101 MHz, CDCl3) δ 156.74, 151.44
(2C), 135.87, 133.56, 128.88 (2C), 128.79 (2C), 123.38, 103.05, 101.41, 63.28, 50.02 (2C), 47.30, 45.30
(2C), 27.23 (2C), 25.01 (2C), 23.90. HRMS m/z 410.2106 [M+H+] (calcd. for C23H28ClN5, 410.2048).
4-([1,4'-Bipiperidin]-1'-yl)-7-(4-fluorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 10b. Brown solid, yield 58
%, Mp 118-120 °C. 1H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H, H2), 7.42 (dd, J = 8.2, 5.5 Hz, 2H, H3’ and
H5’), 7.23 (t, J = 8.6 Hz, 2H, H2’ and H6’), 7.15 (d, J = 3.6 Hz, 1H, H6), 6.74 (d, J = 3.6 Hz, 1H, H5), 5.60
(s, 2H, CH2), 5.11 (d, J = 13.3 Hz, 2H, H2’’ and H6’’), 3.33 (t, J = 12.2 Hz, 2H, H2’’ and H6’’), 3.07 (t, J =
11.5 Hz, 1H, H4’’), 2.93 (br, 4H, 2 x H8’’ and 2 x H12’’), 2.34 (d, J = 12.0 Hz, 2H, H9’’ and H11’’), 2.06 –
1.84 (m, 6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.75 (d, J = 4.9 Hz, 2H, 2 x H10’’). 13C NMR (101 MHz,
CDCl3) δ 162.30 (d, JCF = 246.1 Hz), 156.79, 151.46, 151.42, 133.16 (d, JCF = 3.2 Hz), 129.19 (d, JCF = 8.2
Hz, 2C), 123.27, 115.61 (d, JCF = 21.6 Hz, 2C), 103.05, 101.36, 63.20, 50.08 (2C), 47.24, 45.40 (2C), 27.43
(2C), 25.34 (2C), 24.11. 19F NMR (376 MHz, CDCl3) δ -114.67. HRMS m/z 394.2402 [M+H+] (calcd. for
C23H28FN5, 394.2346).
4-((4-([1,4'-Bipiperidin]-1'-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzonitrile 11b. Brown solid,
yield 52 %, Mp 101-103 °C. 1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H, H2), 7.57 (d, J = 8.1 Hz, 2H, H3’ and
H5’), 7.22 (d, J = 8.0 Hz, 2H, H2’ and H6’), 6.90 (d, J = 3.5 Hz, 1H, H6), 6.53 (d, J = 3.5 Hz, 1H, H5), 5.43
(s, 2H, CH2), 4.84 (d, J = 13.2 Hz, 2H, H2’’ and H6’’), 3.08 (t, J = 12.4 Hz, 2H, H2’’ and H6’’), 2.77 (t, J =
11.3 Hz, 1H, H4’’), 2.68 – 2.57 (br, 4H, 2 x H8’’ and 2 x H12’’), 2.06 (d, J = 11.9 Hz, 2H, H9’’ and H11’’),
1.77 – 1.56 (m, 6H, 2 x H3’’, 2 x H5’’, H9’’ and H11’’), 1.47 (s, 2H, 2 x H10’’). 13C NMR (101 MHz, CDCl3)
δ 156.83, 151.69, 151.52, 142.84, 132.56 (2C), 127.77 (2C), 123.15, 118.55, 111.61, 102.99, 101.94,
15

63.05, 50.14 (2C), 47.53, 45.43 (2C), 27.59 (2C), 25.54 (2C), 24.23. HRMS m/z 401.2448 [M+H+] (calcd.
for C24H28N6, 401.2392).
4-([1,4'-Bipiperidin]-1'-yl)-7-(2,6-dichlorobenzyl)-7H-pyrrolo[2,3-d]pyrimidine 12b. White solid, yield
51 %, Mp 122-124 °C. 1H NMR (400 MHz, CDCl3) δ 8.27 (s, 1H, H2), 7.24 (d, J = 8.0 Hz, 2H, H3’ and H5’),
7.12 (t, J = 8.0 Hz, 1H, H4’), 6.52 (d, J = 3.6 Hz, 1H, H6), 6.25 (d, J = 3.7 Hz, 1H, H5), 5.52 (s, 2H, CH2),
4.73 (d, J = 13.4 Hz, 2H, H2’’ and H6’’), 3.01 – 2.77 (m, 3H, H2’’, H6’’ and H4’’), 2.67 (br, 4H, 2 x H8’’ and
2 x H12’’), 2.04 (d, J = 11.7 Hz, 2H, H9’’ and H11’’), 1.72 (br, 4H, 2 x H3’’ and 2 x H5’’), 1.57 (qd, J = 12.1,
3.6 Hz, 2H, ), 1.40 (s, 2H, 2 x H10’’). 13C NMR (101 MHz, CDCl3) δ 156.63, 151.51, 151.17 (2C), 136.98,
131.93, 130.36, 128.72 (2C), 122.18, 103.01, 100.97, 63.65, 49.93 (2C), 45.14 (2C), 43.10, 26.83 (2C),
24.41 (2C), 23.54. HRMS m/z 444.1716 [M+H+] (calcd. for C23H27Cl2N5, 444.1655).
2.2.3 Pharmacological Part
2.2.3.1 Radioligand Displacement Assays at the H3R
Radioligand displacement assays at the human histamine H3R were performed as published
previously.18 Briefly, membrane preparations of HEK-293 cells stably expressing the human histamine
H3R (20 µg/well) were incubated with the compound and [3H]Nα-methylhistamine (2 nM) in 200 µL of
binding buffer (10 mM MgCl2, 100 mM NaCl and 75 mM Tris/HCl, pH 7.4) for 90 min. Compounds were
tested in duplicates with 11 concentrations in at least three independent experiments. Data was
analyzed with GraphPad Prism 7, using non-linear least squares fit and equation “one site competition”
(representative figures given in the SI). Ki values were calculated according to the Cheng-Prusoff
equation.19 Values are reported as means with the 95% confidence interval. All statistical operations
were performed on the pKi values and converted afterwards to mean Ki values and the 95% confidence
interval. Differences are considered significant if 95% confidence intervals are not overlapping.
2.2.3.2 Radioligand Displacement Assays at the H4R
H4R radioligand displacement assays were performed as described previously.20 Briefly, Sf9 cell
membrane preparations, expressing the human histamine H4R (40 µg/well), were co-incubated with
the compound and [3H]histamine (10 nM) in 200 µL for 60 min. Compounds were tested in duplicates
with at least five concentrations in two independent experiments. Data was analyzed with GraphPad
Prism 7.
3 Results and Discussion
16

The first synthesized set (compounds 3a-3c and 4a-4c) consisted of compounds bearing a
morpholine group instead of the lipophilic core (R) that was connected to the pyrrolo[2,3-d]pyrimidine
via an ethyl linker. All compounds displayed marked affinity at the H3R without H4R affinity (Ki value
> 10 µM). The general introduction of the morpholine group instead of a lipophilic core improved
binding affinity slightly, when compared to compound V (Ki value 126 nM), with Ki values below 50 nM
for derivatives 4a and 4b, although results were not significant. By comparing binding affinities of series
3 to 4 (Table 1) the pharmacodynamic superiority of 1,4-bipiperidine moieties (series 4) as basic
warhead (Y), compared to 4-(piperidin-4-yl)morpholines (series 3), was once more verified.16 The
exchange towards the 1,4-bipiperidine resulted in a significant increase of binding affinity.
Derivatization at position 2 (X) gave no significant difference between chlorinated compounds or the
unsubstituted central core but revealed a pronounced loss of affinity for the aminated compounds (3c,
4c). Due to these findings, only compounds 4a and 4b were further derivatized.
17

Table 1: Affinities at the H3R of compounds 3a-3c, 4a-4c, 9a-12a and 9b-12b.
Substitution pattern
Y
Compound
Ki (nM)a)
[95% CI nM]
317
X
R
b)
3a
3b
Cl
O
Morph-CH2
[135;743]
374
[162;865]
H
NH2
Cl
O
Morph-CH2
Morph-CH2
Morph-CH2
Morph-CH2
Morph-CH2
Ph-4-Clc)
848
[386;1866]
3c
O
22
[11;45]
4a
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
31
[16;62]
4b
H
121
[49;301]
4c
NH2
Cl
42
[14;127]
9a
26
[15;46]
9b
H
Ph-4-Cl
30
[11;81]
10a
10b
11a
11b
12a
12b
Cl
Ph-4-Fd)
35
[10;120]
H
Ph-4-F
18
[5;66]
Cl
Ph-4-CNe)
Ph-4-CN
7
H
[4;11]
54
[25;116]
Cl
Ph-2,6-diClf)
6
H
Ph-2,6-diCl
[2;19]
a)Ki values were measured at the human histamine H3R. Membrane preparations of HEK-293T cells expressing the H3R
(20 µg/well) were incubated with the compound of interest and [3H]Nα-methylhistamine (2 nM) in 200 µL for 90 min.
b)Morph-CH2, morpholinomethyl. c) Ph-4-Cl, 4-chlorophenyl. d) Ph-4-F, 4-fluorophenyl. e) Ph-4-CN, 4-cyanophenyl. f) Ph-2,6-
diCl, 2,6-dichlorophenyl.
In compounds 9a-12a the 4-([1,4'-bipiperidin]-1'-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine
moiety was connected to differently substituted benzyl moieties. Inspired by the substitution pattern
18

of pitolisant, the first ligand displayed a 4-chlorophenyl residue, which was exchanged by several other
electron-withdrawing substituted benzyl derivatives. The compounds showed high affinity at the H3R,
without affinity at the H4R (Ki value > 10 µM) and no significant changes in affinity, based on the
derivatization pattern of the benzyl (Table 1).
For the compounds 9b-12b without chlorination on the heterocyclic moiety affinity at the H3R
increased by increasing electron-withdrawing properties of the aromatic core (Table 1), while no
compound showed considerable affinity at the H4R (Ki values > 10 µM). For compound 11b the increase
of binding affinity at the H3R was significant when compared to those of 4b (morpholinomethyl as R)
or 9b, the compound with the weakest electron-withdrawing properties. Furthermore, compound 12b
is the only compound where dechlorination led to a significant increase of binding affinity compared
to the chlorinated compound (12a). When compared to compound V the introduction of a cyano
moiety (11b) or a 2,6-dichlorobenzyl (12b) increased affinity significantly with Ki values of 7 and 6 nM,
respectively. Although functional studies were not performed on the scaffold of pyrrolo[2,3-
d]pyrimidines, the evidence of many non-imidazole ligands being H3R antagonist or inverse agonists
(aplysamine-1, conessine and pitolisant6,7,11) could rationalize an in vivo evaluation of these novel
compounds as H3R antagonists/inverse agonists.
Based on the lead structure the presented compounds allow for an initial structure-activity
relationship evaluation for the class of pyrrolo[2,3-d]pyrimidine as novel H3R scaffolds.
The 1,4-bipiperidine motif in position 4 seems to be crucial for high affinity towards the H3R (compare
series 3 and 4). The introduction of a 2-morpholinoethyl instead of a 2-naphthylmethyl moiety in
position 7 (compounds 3a-3c, 4a-4c) improved binding affinity not significantly compared to
compound V. The introduction of an amine group at position 2 leads to a loss in affinity (series c,
significant for 4a to 4c). For a given structure, with or without chloro substitution in 2-position did not
result in any significant affinity changes (compare 3a to 3b, 4a to 4b, etc.), except for the most active
compound 12b. The introduction of substituted benzyl moieties at 7-position did not further increase
binding affinity, if chlorinated at 2-position (compounds 9a-12a) and derivatization pattern of the
benzyl did not influence affinity. For compounds without chloro or amino substitution at 2-position of
the heterocycle (9b-12b) affinity was increased by increasing electron-withdrawing abilities of the
substitution pattern, with the most active compounds (11b and 12b) displaying a significantly higher
affinities than that of compound V.
4 Conclusion
19

Within this study we were able to synthesize 14 novel pyrrolo[2,3-d]pyrimidines that adhere to the
general pharmacophore of H3R ligands. The 1,4-bipiperidine motif as basic centre has proven itself
highly effective for designing H3R ligands with superior binding affinity. The arbitrary, lipophilic region
was best modulated by benzyl residues with electron-withdrawing groups. In vivo evaluation of these
nature inspired synthetic pyrrolo[2,3-d]pyrimidines will be highly interesting, as the potential anti-
inflammatory properties of this class may have synergistic effects with the human H3R modulation21
on cognitive impairments in neurodegenerative diseases. By that approach drug development of CNS
disorders may be complemented by a new class of active agents and natural compounds have once
more proven their versatility within the drug research and development community.
Acknowledgement: Support by the DFG with GRK2158, INST 208/664-1 and by FONDECYT project N°
11180292 is greatly acknowledged.
Additional analytic information can be found in the Supplemental Material at www…..
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21

Figure 4: Representative figures of the radioligand displacement assays at the human H3R for compounds 3a-3c and 4a-4c.
Figure displays specific CPM as means with respective standard error of the mean (SEM), non-normalized.
Figure 5: Representative figures of the radioligand displacement assays at the human H3R for compounds 9a-12a and 9b-
12b. Figure displays specific CPM as means with respective standard error of the mean (SEM), non-normalized.
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Graphical abstract
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